Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated <i>MUTYH</i> variants

Functional characterization of genetic variants has the potential to advance the field of precision medicine by enhancing the efficacy of current therapies and accelerating the development of new approaches to combat genetic diseases. MUTYH is a DNA repair enzyme that recognizes and repairs oxidatively damaged guanines [8-oxoguanine (8-oxoG)] mispaired with adenines (8-oxoG·A). While some mutations in the MUTYH gene are associated with colorectal cancer, most MUTYH variants identified in sequencing databases are classified as variants of uncertain significance. Convoluting clinical classification is the absence of data directly comparing homozygous versus heterozygous MUTYH mutations. In this study, we present the first effort to functionally characterize MUTYH variants using precision genome editing to generate heterozygous and homozygous isogenic cell lines. Using a MUTYH-specific lesion reporter in which we site-specifically incorporate an 8-oxoG·A lesion in a fluorescent protein gene, we measure endogenous MUTYH enzymatic activity and classify them as pathogenic or benign. Further, we modify this reporter to incorporate the MUTYH repair intermediate (8-oxoG across from an abasic site) and validate it with co-immunoprecipitation experiments to demonstrate its ability to characterize the mechanism by which MUTYH mutants are defective at DNA repair.